Formaldehyde neutral buffer solution

The cells were harvested by dispase treatment, collected into tubes, and centrifuged, and the pellets were suspended in DMEM. A total of 1,000,000 hESCs were injected into the rear leg muscle or thigh muscle of a SCID (C.B-17/lcr-scid/scidJcl) mouse (CLEA Japan, Tokyo, Japan). Seven weeks after injection, tumors were dissected, weighed, and fixed with 10% formaldehyde Neutral Buffer Solution (Nacalai tesque, Kyoto, Japan). Paraffin-embedded tissue was sliced and stained with hematoxylin and eosin. All animal experiments were conducted in accordance with the guidelines for animal experiments of the National Institute of Biomedical Innovation, Health, and Nutrition, Osaka, Japan.

HeLa cells were grown on uncoated glass coverslips and transfected with pcDNA3/cmHNF-4 and pcDNA3/FLAG-cmSHP using Polyethylenimine Max (Polysciences, Inc.). At 24 h after transfection, the cells were fixed with 10% Formaldehyde Neutral Buffer Solution (Nacalai Tesque) for 10 min, mounted on glass slides, and observed with a BZ-8100 fluorescence microscope (Keyence) or an LSM510 confocal laser-scanning microscope (Zeiss). To detect HNF-4 and FLAG-SHP, the cells were incubated with an anti-HNF-4 antibody and anti-DYKDDDDK antibody overnight at 4 °C, washed three times with 0.5% BSA in PBS, and incubated with Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Thermo Scientific) for 1 h at room temperature. To detect nuclei using a fluorescence microscope, the cells were washed twice with PBS and stained with 1 μg/ml Hoechst 33258 (Thermo Scientific) solution for 10 min at room temperature. The cells were then washed with H₂O, mounted on a slide, and observed under the microscope.

HE staining was performed on the preserved cell sheets and human corneal limbal tissues to examine the degree of stratification and morphology of the cell sheets. One quarter of the cell sheet and the corneal limbal tissues were fixed with 10% formaldehyde neutral buffer solution (Nacalai Tesque) at room temperature overnight. After washing with distilled water, the cell sheets and corneal limbal tissues were embedded in paraffin and processed into 3-μm-thick sections. The sections were deparaffinized and hydrated, and were stained with HE. Microphotographs were taken with BIOREVO BZ-9000 (KEYENCE, Co.), and the morphology was examined.

GalNAc4S-6ST-knockout mice were generated as described previously [13] (link). Mice used in the CCl₄-induced fibrosis study had been back-crossed to BALB/c mice for more than 10 generations. Mice used for preparation of hepatic stellate cell (HSC) had been back-crossed to C57BL/6 N mice for more than 10 generations. Mice were maintained in 12-hour light/12-hour dark cycles with free access to food and water in a pathogen-free room at the Laboratory Animal Research Center, Aichi Medical University. Mice (7-to-8-week-old females) were injected intraperitoneally with carbon tetrachloride (CCl₄), which was diluted 2:5 in mineral oil (Sigma) at a dose of 2.0 ml/kg (as CCl₄) of body weight twice a week for a total of 9 injections. Control mice were injected with mineral oil alone. Number of mice used for analysis was indicated under the legend of each figure. Mice were killed 2 days, 3 weeks, 6 weeks or 9 weeks after the last CCl₄ injection, and blood and liver samples were obtained. The liver was either fixed with 10% formaldehyde neutral buffer solution (Nakalai Tesque, Kyoto, Japan) for histological examination or used for extraction of RNA, analysis of glycosaminoglycans and measurement of the content of hydroxyproline. All animal procedures were approved by Animal Research Committee of Aichi Medical University.

The distal half of the colon tissue was harvested, fixed with 10% (w/v) Formaldehyde Neutral Buffer Solution (Nacalai, Kyoto, Japan) overnight, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E) and with Periodic acid–Schiff (PAS). Crypt-cell proliferation and tight-junction were determined by immunohistochemistry with anti-Ki67 antibody (#652402, Biolegend, San Diego, CA) and anti-ZO-1 antibody (#GTX108592, Genetex, Irvine, CA), respectively. Apoptosis was detected by TUNEL assays, using the In Situ Cell-Death Detection Kit, peroxidase (POD) (Roche, Basel, Switzerland). For Alcian blue/PAS staining, the distal colon was harvested, submerged in Carnoy’s solution (Wako, Japan) at 4 °C for 2 h, and placed into 100% ethanol. The fixed colon tissues were embedded in paraffin, cut into 5 μm sections, and stained with Alcian blue/PAS. Microscopic images of the organoid cultures were obtained with a BZ-X700 (Keyence, Osaka, Japan).

The virus stock was diluted 10-fold (1:10–1:10⁶) in FBS-free minimal essential medium (MEM). Diluted viruses (250 μL) were inoculated into the Vero cell monolayer in a 24-well plate and incubated at 37 °C for 2 h. The cells were overlaid with 500 μL MEM (Thermo Fisher Scientific, Cat #11935046) supplemented with 3% FBS and 1.5% carboxymethylcellulose sodium salt (Sigma-Aldrich, Cat# C4888-500G), and the plate was incubated at 37 °C for three days. The cells were washed three times with phosphate-buffered saline (PBS) (+) and fixed with 10% formaldehyde neutral buffer solution (Nacalai Tesque, Cat# 37152-51) for 20 min. After permeabilization with 1% Triton X-100 (Nacalai Tesque, Cat# 35501-15) in PBS (−) for 5 min, cells were incubated with mouse anti-flavivirus NS3 monoclonal antibody (34B1) [60 (link)] at 37 °C for 60 min. After washing with PBS (−), cells were incubated with goat anti-mouse IgG (H+L)-HRP (KPL, Cat# 074-1806) at 37 °C for 60 min. The foci of the infected cells were visualized using a Peroxidase Stain 3,3′-diaminobenzidine (DAB) Kit (Nacalai Tesque, Cat# 25985-50) prepared in the Metal Enhancer for DAB Stain (Nacalai Tesque, Cat# 07388-24).

Preserved hOEC sheets were trypsinized with 0.25% trypsin-EDTA and resuspended in KCM. For hOEC sheets before preservation and those preserved in HBSS + ebselen, the cell suspensions were seeded to 5% of a single cell sheet on NIH-3T3 feeder layers in 100-mm cell culture dishes (BD Falcon™). hOEC sheets preserved in other media were seeded over the whole cell suspension on NIH-3T3 feeder layers in 100-mm cell culture dishes. The seeding cells were cultivated in KCM for 10 days at 37 °C and 5% CO₂. Colonies were fixed with a 10% formaldehyde neutral buffer solution (Nacalai Tesque), and stained with 2% rhodamine. The number of colonies was counted and has been represented by bar graphs.